Composition of restricted cancer cells which produce cancer cell proliferation suppressive materials, and uses thereof

ABSTRACT

Compositions of matter are described which contain restricted cancer cells. When so restricted, the cells produce an unexpectedly high amount of material which suppresses cancer cell proliferation. The phenomenon crosses cancer type and species lines. Processes for making these compositions, and their use, are also described.

RELATED APPLICATION

[0001] This application is a continuation in part of allowed patentapplication Ser. No. 08/745,063, filled on Nov. 7, 1996, which is acontinuation-in-part of co-pending application Ser. No. 08/625,595,filed Apr. 3, 1996 now abandoned. Both are incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the restriction of theproliferation of cancer cells to produce material which suppressesproliferation of unrestricted cancer cells. The structures which are onefeature of the invention can be used “as is,” or to produce materialsuch as concentrates with a minimum approximate molecular weight, whichalso have an anti-proliferative effect on cancer.

BACKGROUND AND PRIOR ART

[0003] The encapsulation of various biological materials in biologicallycompatible materials, which is well documented in the literature, is atechnique that has been used for some time, albeit with limited success.Exemplary of the art are U.S. Pat. No. 5,227,298 (Weber, et al.); U.S.Pat. No. 5,053,332 (Cook, et al.); U.S. Pat. No. 4,997,443 (Walthall, etal.); U.S. Pat. No. 4,971,833 (Larsson, et al.); U.S. Pat. No. 4,902,295(Walthall, et al.); U.S. Pat. No. 4,798,786 (Tice, et al.); U.S. Pat.No. 4,673,566 (Goosen, et al.); U.S. Pat. No. 4,647,536 (Mosbach, etal.); U.S. Pat. No. 4,409,331 (Lim); U.S. Pat. No. 4,392,909 (Lim); U.S.Pat. No. 4,352,883 (Lim); and U.S. Pat. No. 4,663,286 (Tsang, et al.).Also of note is U.S. Pat. No. 5,643,569 to Jain, et al., incorporated byreference herein. Jain, et al. discuss, in some detail, theencapsulation of islets in various biocompatible materials. Isletsproduce insulin, and the use of the materials disclosed by Jain, et al.in the treatment of diabetes is taught therein.

[0004] The Jain, et al. patent discusses, in some detail, the priorapproaches taken by the art in transplantation therapy. These aresummarized herein as well.

[0005] Five major approaches to protecting the transplanted tissue fromthe host's immune response are known. All involve attempts to isolatethe transplanted tissue from the host's immune system. Theimmunoisolation techniques used to date include: extravascular diffusionchambers, intravascular diffusion chambers, intravascularultrafiltration chambers, microencapsulation, and macroencapsulation.There are many problems associated With methods of the prior art,including a host fibrotic response to the implant material, instabilityof the implant material, limited nutrient diffusion acrosssemi-permeable membranes, secretagogue and product permeability, anddiffusion lag-time across semi-permeable membrane barriers.

[0006] For example, a microencapsulation procedure for enclosing viablecells, tissues, and other labile membranes within a semipermeablemembrane was developed by Lim in 1978. (Lim, Research report to DamonCorporation (1978)). Lim used microcapsules of alginate and polyL-lysine to encapsulate the islets of Langerhans. In 1980, the firstsuccessful in vivo application of this novel technique in diabetesresearch was reported (Lim, et al., Science 210: 908 (1980)). Theimplantation of these microencapsulated islets of Langerhans resulted insustaining a euglycemic state in diabetic animals. Other investigators,however, repeating these experiments, found the alginate to cause atissue reaction and were unable to reproduce Lim, et al.'s results(Lamberti, et al. Applied Biochemistry and Biotechnology 10: 101 (1984);Dupuy, et al., J. Biomed. Material and Res. 22: 1061 (1988); Weber, etal., Transplantation 49: 396 (1990); and Doon-shiong, et al.,Transplantation Proceedings 22: 754 (1990)). The water solubility ofthese polymers is now considered to be responsible for the limitedstability and biocompatibility of these microcapsules in vivo (Dupuy, etal., supra, Weber et al., supra, Doon-shiong, et al., supra, andSmidsrod, Faraday Discussion of Chemical Society 57: 263 (1974)).

[0007] Iwata et al., (Iwata, et al. Jour. Biomedical Material and Res.26: 967 (1992)) utilized agarose for microencapsulation of allogeneicpancreatic islets and discovered that it could be used as a medium forthe preparation of microbeads. In their study, 1500-2000 islets weremicroencapsulated individually in 5% agarose and implanted intostreptozotocin-induced diabetic mice. The graft survived for a longperiod of time, and the recipients maintained normoglycemiaindefinitely.

[0008] Their method, however, suffers from a number of drawbacks. It iscumbersome and inaccurate. For example, many beads remain partiallycoated and several hundred beads of empty agarose form. Additional timeis thus required to separate encapsulated islets from empty beads.Moreover, most of the implanted microbeads gather in the pelvic cavity,and a large number of islets in completely coated individual beads arerequired to achieve normoglycemia. Furthermore, the transplanted beadsare difficult to retrieve, tend to be fragile, and will easily releaseislets upon slight damage.

[0009] A macroencapsulation procedure has also been tested.Macrocapsules of various different materials, such aspoly-2-hydroxyethyl-methacrylate, polyvinylchloride-c-acrylic acid, andcellulose acetate were made for the immunoisolation of islets ofLangerhans. (See Altman, et al., Diabetes 35: 625 (1986); Altman, etal., Transplantation: American Society of Artificial Internal Organs 30:382 (1984); Ronel, et al., Jour. Biomedical Material Research 17: 855(1983); Klomp, et al., Jour. Biomedical Material Research 17: 865-871(1983)). In all these studies, only a transitory normalization ofglycemia was achieved.

[0010] Archer, et al., Journal of Surgical Research 28: 77 (1980), usedacrylic copolymer hollow fibers to temporarily prevent rejection ofislet xenografts. They reported long-term survival of dispersed neonatalmurine pancreatic grafts in hollow fibers which were transplanted intodiabetic hamsters. Recently Lacy, et al., Science 254: 1782-1784 (1991)confirmed their results, but found the euglycemic state to be atransient phase. They found that when the islets are injected into thefiber, they aggregate within the hollow tube with resultant necrosis inthe central portion of the islet masses. The central necrosis precludedprolongation of the graft. To solve this problem, they used alginate todisperse the islets in the fiber. However, this experiment has not beenrepeated extensively. Therefore, the membrane's function as an islettransplantation medium in humans is questionable.

[0011] The Jain, et al. patent discussed reports that encapsulatingsecretory cells in a permeable, hydrophilic gel material results in afunctional, non-immunogenic material, that can be transplanted intoanimals, can be stored for long lengths of time, and is therapeuticallyuseful in vivo. The macroencapsulation of the secretory cells provided amore effective and manageable technique for secretory celltransplantation.

[0012] The patent does not discuss at any length the incorporation ofcancer cells. A survey of the literature on encapsulation of cellsreveals that, following encapsulation, cells almost always produce lessof materials than they produce when not encapsulated. See Lloyd-George,et al., Biomat. Art. Cells & Immob. Biotech. 21(3): 323-333 (1993);Schinstine, et al., Cell Transplant 4(1): 93-102 (1995); Chicheportiche,et al., Diabetologica 31:54-57 (1988); Jaeger, et al., Progress In BrainResearch 82:41-46 (1990); Zekorn, et al., Diabetologica 29:99-106(1992); Zhou, et al., Am. J. Physiol. 274: C1356-1362 (1998); Darquy, etal., Diabetologica 28:776-780 (1985); Tse, et al., Biotech. & Bioeng.51:271-280 (1996); Jaeger, et al., J. Neurol. 21:469-480 (1992);Hortelano, et al., Blood 87(12): 5095-5103 (1996); Gardiner, et al.,Transp. Proc. 29:2019-2020 (1997). None of these references deal withthe incorporation of cancer cells into a structure which entraps themand restricts their growth, but nonetheless permit diffusion ofmaterials into and out of the structure.

[0013] One theory relating to the growth of cancerous masses likens suchmasses, e.g., tumors, to normal organs. Healthy organs, e.g. the liver,grow to a particular size, and then grow no larger; however, if aportion of the liver is removed, it will regenerate to a certain extent.This phenomenon is also observed with tumors. To summarize, it has beennoted that, if a portion of a tumor is removed, the cells in theremaining portion of the tumor will begin to proliferate very rapidlyuntil the resulting tumor reaches a particular size, after whichproliferation slows down, or ceases. This suggests that there is someinternal regulation of cancer cells.

[0014] The invention, which will be seen in the following disclosure,shows that when cancer cells are restricted by being entrapped, theirproliferation is halted, and they produce unexpectedly high amounts ofmaterial which, when applied to non-restricted cancer cells, inhibitsthe proliferation of these non-restricted cancer cells. The ability toretard proliferation of cancer cells has been a goal of oncology sinceits inception. Hence, the therapeutic usefulness of this invention willbe clear and will be elaborated upon herein. The material produced doesnot appear to be limited by the type of cancer cell used, nor by theanimal species from which the cancer cells originate. Further, theeffect does not appear to be species specific, as restricted cells froma first species produce material which inhibits proliferation ofunrestricted cells from a second species. Also, the effect does notappear to be specific to the type of cancer, as restricted cells from afirst cancer type produce material which inhibits proliferation ofunrestricted cells from another cancer type.

[0015] Nor does the effect appear to require an immune response. Theantiproliferative effect is seen in in vitro systems, where no immunecells are used. Hence the antiproliferative effect cannot be attributedto classical immunological responses.

[0016] Thus, a preferred embodiment of the invention relates to acomposition of matter having a biocompatible, proliferation-restrictive,selectively-permeable structure. The structure restricts cancer cellswhich then produce more of a material which suppresses cancer cellproliferation compared to an equal number of the same cancer cells whenunrestricted.

[0017] Another preferred embodiment of the present invention relates toa process for preparing a biocompatible, proliferation-restrictive,selectively-permeable structure, by forming a structure by contactingcancer cells with biocompatable, proliferation-restrictive matter toform the structure, and culturing the structures for a sufficient periodof time to restrict the cancer cells such that they produce a materialwhich suppresses cancer cell proliferation compared to an equal numberof unrestricted cancer cells of the same cancer type.

[0018] Yet another preferred embodiment relates to a method ofincreasing the production of material that suppresses cancer cell growthby a cancer cell, comprising restricting cancer cells in astructure-forming material to form a biocompatable,selectively-permeable, proliferation-restrictive structure and culturingthe cancer cells until they are restricted and produce the material.

[0019] It has also been found that a powerful antiproliferative effectcan be achieved by subjecting conditioned medium obtained by culturingthe structures of the invention in culture medium to filtration. Theresulting concentrates have extremely strong anti-proliferative effects.

[0020] The material, the conditioned medium, and/or the concentratesderived therefrom may also be useful for inducing the production of theanti-proliferative material by other non-restricted cancer cells.

[0021] These, and other features of the invention, will be seen from thedisclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0022] This example, and those which follow, employ RENCA cells. Theseare spontaneous renal adenocarcinoma cells of BALB/C mice, which arewidely available, having been maintained in both in vitro cultures andin vivo. See Franco, et al., Cytokine Induced Tumor Immunogenecity,181-193 (1994).

[0023] Samples of frozen RENCA cells were thawed at 37° C., and thenplaced in tissue culture flasks containing Dulbecco's Modified Medium(D-MEM), which had been supplemented with 10% bovine serum, penicillin(100 u/ml) and streptomycin (50 ug/ml), to give what will be referred toas “complete medium” hereafter.

[0024] Cells were grown to confluence, and then trypsinized, followed bywashing with Hank's Balanced Salt Solution, and then with the completemedium referred to supra.

[0025] In order to determine if the RENCA cells produced tumorsefficiently, two BALB/C mice were injected, intraperitoneally, with 10⁶of these cells. The mice were observed, over a 3-4 week period.Clinically, they appeared healthy for the first two weeks, and exhibitednormal activity. Thereafter, the clinical manifestations of cancerbecame evident. One mouse died after 23 days, and the second, after 25days. Following death, the mice were examined, and numerous tumors ofvarious size were observed. Some of the tumors exhibited hemorrhaging aswell.

[0026] A sample of one tumor, taken from one of the mice, was fixed in10% formalin for later histological examination.

EXAMPLE 2

[0027] Following the showing that the RENCA cells did grow in vivo,studies were carried out to determine if these cells grew whenrestricted in the structure of the invention.

[0028] RENCA cells were grown to confluency, as described supra,trypsinized, and washed, also as described above. Samples of between60,000 and 90,000 cells were then prepared. The cells were thencentrifuged, at 750 RPMs, and fluid was removed. The cells were thensuspended in solutions of 1% atelocollagen, in phosphate buffered salinesolution, at a pH of 6.5.

[0029] A 1% solution of low viscosity agarose was prepared in minimalessential medium (MEM), maintained at 60° C., and then 100 ul of thiswas added to the suspension of RENCA cells and atelocollagen, describedsupra. The materials were then transferred, immediately, as a singlelarge droplet, into sterile, room-temperature mineral oil. The mixtureformed a single, smooth, semi-solid bead. This procedure was repeated toproduce a number of beads.

[0030] After one minute, the beads were transferred to complete medium,as described supra, at 37° C. The beads were then washed three times inMinimal Essential Medium (MEM) containing the antibiotics listed supra.The beads were then incubated overnight at 37° C., in a humidifiedatmosphere of air and 5% CO₂. Following the incubation the beads, nowsolid, were transferred to a sterile spoon which contained 1 ml of 5%agarose in MEM. Beads were rolled in the solution 2-3 times to uniformlycoat them with agarose. The beads were transferred to mineral oil beforethe agarose solidified, to yield a smooth outer surface. After 60seconds, the beads were washed, five times, with complete medium at 37°C. to remove the oil. Overnight incubation (37° C., humidifiedatmosphere of air, 5% CO₂) followed. These RENCA containing beads wereused in the experiments which follow.

EXAMPLE 3

[0031] Prior to carrying out in vivo investigations, it was necessary todetermine if the RENCA cells would grow in beads prepared in the mannerdescribed supra.

[0032] To do this, beads prepared as discussed in example 2 wereincubated in the medium described in example 2, for a period of threeweeks, under the described conditions. Three of the beads were then cutinto small pieces, and cultured in standard culture flasks, affordingdirect contact with both the flask and culture medium.

[0033] Observation of these cultures indicated that the cells grew andformed standard RENCA colonies. This indicated that the cells hadremained viable in the beads.

EXAMPLE 4

[0034] In vivo experiments were then carried out. In these experiments,the beads were incubated for seven days, at 37° C. Subject mice thenreceived bead transplants. To do this, each of four mice received amidline incision, carried through intraperitoneally. Three beads, eachof which contained 60,000 RENCA cells were transplanted. Incisions werethen closed (two-layer closure), using an absorbable suture. The fourmice (BALB/C) were normal, male mice, weighing between 24-26 grams, andappeared to be healthy. Two sets of controls were set up. In the firstset, two mice received three beads containing no RENCA cells, and in thesecond, two mice were not treated with anything.

[0035] Three weeks after the implantation, all of the mice receivedintraperitoneal injections of 10⁶ RENCA cells. Eighteen days later, onecontrol mouse died. All remaining mice were then sacrificed, andevaluated for the presence or absence of tumor.

[0036] Control mice showed numerous tumors, while the mice whichreceived the implants of bead-encapsulated cells showed only isolatedsmall nodules throughout the cavity.

[0037] These encouraging results suggested the design of the experimentsset forth in the following example.

EXAMPLE 5

[0038] In these experiments, established cancers were simulated byinjecting RENCA cells under one kidney capsule of each of six BALB/Cmice. Fifteen days later, mice were divided into two groups. Thethree-mice in the first group each received three beads, as described inexample 4, supra. The second group (the control group) received beadswhich did not contain RENCA cells.

[0039] For the initial 4-5 days, mice which had received RENCA cellcontaining implants looked lethargic, and their fur had become spiky.Thereafter, they returned to normal. The control group remainedenergetic, with no change in condition of fur.

[0040] Ten days after implantation (25 days after injection of RENCAcells), however, the control mice became sluggish and exhibiteddistended abdomens. One of the three control mice died at fourteen daysfollowing bead transplantation. Sacrifice of the mice followed.

[0041] The body cavities of the control mice showed profusehemorrhaging, with numerous tumors all over the alimentary canal, liver,stomach and lungs. All organs of the abdominal cavity had becomeindistinguishable due to rampant tumor growth. The mice which hadreceived beads with encapsulated RENCA cells, however showed nohemorrhaging, and only a few nodules on the alimentary canal. Inaddition, comparison of test and control groups showed that in the testgroup, nodules had not progressed beyond their initial growth under thekidney capsule and before macrobead implantation.

EXAMPLE 6

[0042] In vitro, freely inoculated RENCA cell growth is inhibited whensuch cells are incubated along with macrobead encapsulated RENCA cells.A further set of experiments was carried out to determine if this effectwas observable with other cells.

[0043] An adenocarcinoma cell line, i.e., MMT (mouse mammary tumor), wasobtained from the American Type Culture Collection. Encapsulated MMTcells were prepared, as described, supra with MMT cells, to producebeads containing 120,000 or 240,000 cells per bead. Followingpreparation of the beads, they were used to determine if they wouldinhibit proliferation of RENCA cells in vitro. Specifically, twosix-well petri plates were prepared, via inoculation with 1×10⁴ RENCAcells per well, in 4 ml of medium. In each plate, three wells served ascontrol, and three as test. One of the three control wells in each platereceived one empty bead. Each of the other wells received either two orthree empty beads. The second set of wells was treated similarly, withwells receiving one, two or three beads containing 120,000 or 240,000MMT cells. Wells were incubated at 37° C. for one week, after whichRENCA cells were trypsinized, washed, and counted, using ahemocytometer. Results are shown in Table 1: TABLE 1 DISH #2 DISH #1 #of cells # of cells retrieved after one week retrieved after one weekControl Control (Empty 120,000 (Empty 240,000 Well# macrobead) MMT cellsMacrobead) MMT cells 1 2.4 × 10⁵  1.4 × 10⁵ 2.8 × 10⁵ 1 × 10⁵ 2 2.0 ×10⁵  1.2 × 10⁵ 3.6 × 10⁵ 7 × 10⁴ 3 4.4 × 10⁵ 1.25 × 10⁵ 2.5 × 10⁵ 9 ×10⁴

EXAMPLE 7

[0044] Following the results in example 6, the same experiments wascarried out using 1×10⁴ MMT cells as the inoculant (i.e., the freecells) rather than RENCA cells. The experiment was carried out preciselyas example 6. Results are set forth in Table 2 below. TABLE 2 DISH #1DISH #2 Control 120,000 Control 240,000 (Empty MMT cells in (Empty MMTcells in Well# macrobead) macrobeads Macrobead) macrobeads 1 3.1 × 10⁶1.6 × 10⁶ 2.8 × 10⁶ 1.3 × 10⁶ 2 3.3 × 10⁶ 1.0 × 10⁶ 2.6 × 10⁶ 1.1 × 10⁶3 3.0 × 10⁶ 6.0 × 10⁵ 2.8 × 10⁶ 5.0 × 10⁵

[0045] These results encouraged an in vivo experiment. This is presentedin example 8.

EXAMPLE 8

[0046] The mouse mammary tumor cell line (MMT) described supra was used.Using the protocols set forth, supra, implants were prepared whichcontained 120,000 cells per bead, and 240,000 cells per bead.

[0047] The experimental model used was the mouse model, supra.Twenty-two mice were divided into groups of 4 (control), 9 and 9. Thefirst group, i.e., the controls, were further divided into three groups:two received implants of one empty bead, one received two empty beads,and one received three empty beads.

[0048] Within experimental Group A (9 animals), the beads contained120,000 cells, while in experimental Group B, the beads contained240,000 cells. Within Groups A and B, there were three subdivisions,each of which contained three mice. The subgroups received one, two, orthree beads containing MMT cells.

[0049] For the first few days, the mice in Groups A and B werelethargic, with spiky hair. This persisted for about five days, afterwhich normal behavior was observed. Twenty-one days followingimplantation, all animals received injections of 40,000 RENCA cells.

[0050] After another twenty days, the control mice exhibited distendedabdomens, and extremely spiky hair. One control mouse died twenty-fivedays following injection, while the remaining control mice appearedterminal. All mice were sacrificed, and tumor development was observed.These observations are recorded in Table 3 infra: TABLE 3 NUMBER OFEXPERIMENT- MACROBEADS EXPERIMENTAL AL IN MICE CONTROL GROUP A GROUP B 1++++ − − 1 ++++ − − 1 + ++ 2 ++++ − − 2 − − 2 ++ ++ 3 ++++ − − 3 − − 3 −+++

[0051] These results show that, of eighteen mice treated, thirteenshowed no disease. Of the mice in Group A, one mouse exhibited a fewsmall nodules (+), and another mouse showed a few tumors (++).

[0052] Within Group B, one mouse which had received one bead, and onemouse which received two beads showed a few tumors, entangled withintestine. One of the mice which received three beads had developed alarge solid tumor and was apparently very sick (+++). All control micehad numerous tumors (++++). The results showed that the encapsulatedmouse mammary tumor cells inhibited tumor formation.

EXAMPLE 9

[0053] As suggested, supra, the practice of the invention results in theproduction of material which inhibits and/or prevents tumor cellproliferation. This was explored further in the experiment whichfollows.

[0054] Additional beads were made, as described supra in example 2,except that atelocollagen was not included. Hence, these beads areagarose/agarose beads. RENCA cells, as described, supra, wereincorporated into these beads, again as described supra.

[0055] Two sets of three six-well plates were then used as control andexperimental groups. In the control group, wells were filled with 4 mlof RPMI complete medium (10% fetal calf serum and 11 ml/l ofpenicillin). Each control group well was then inoculated with 10,000RENCA cells.

[0056] In the experimental group, the RPMI complete medium wasconditioned, by adding material secured by incubating ten RENCAcontaining beads (120,000 cells per bead), in a 35×100 mm petri platecontaining 50 ml of the RPMI complete medium. Following five days ofincubation, medium was collected from these plates, and 4 ml of it wasplaced in each test well. These wells were then inoculated with 10,000RENCA cells in each.

[0057] All plates (both control and experimental) were incubated at 37°C. for five days. Following the incubation period, cells weretrypsinized, washed, pooled, and counted using a hemocytometer. Theresults are shown in Table 4: TABLE 4 RENCA CELLS TEST RENCA CELLS WITHCONDITIONED WELL # WITH CONTROL MEDIUM MEDIUM 1 7 × 10⁵   3 × 10⁵ 2 8 ×10⁵ 2.5 × 10⁵ 3 7 × 10⁵ 3.4 × 10⁵

[0058] These results show that the cells, when restricted in, e.g., thebeads of the examples, produced some material which resulted insuppression of tumor cell proliferation.

EXAMPLE 10

[0059] The experiment set forth supra showed that RENCA cell growth, inconditioned medium, was about half the growth of the cells in controlmedium. The experiments set forth herein examined whether thesuppression of proliferation would continue after the conditioned mediumwas frozen.

[0060] RENCA conditioned medium was prepared by incubating ten RENCAcontaining beads for five days. Incubation was in 35×100 mm petriplates, with 50 ml RMPI complete medium, at 37° C. Following theincubation, the medium was collected and stored at −20° C. Conditionedmedium was prepared by incubating MMT (mouse mammary tumor) cellcontaining beads. The beads contained 240,000 cell per bead; otherwiseall conditions were the same.

[0061] Frozen media were thawed at 37° C., and then used in thefollowing tests. Three six-well plates were used for each treatment,i.e., (i) RMPI control medium, (2) RENCA frozen conditioned medium, and(3) MMT frozen conditioned medium. A total of 4 ml of medium wasdispensed into each well. All wells were then inoculated with 10,000RENCA cells, and incubated at 37° C., for five days. Followingincubation, two plates of samples were taken from each well,trypsinized, washed, pooled, and counted in a hemocytometer. At eightdays, the remaining three plates of each well were tested in the sameway.

[0062] Results follow: TABLE 5 FROZEN FROZEN CONTROL CONDITIONEDCONDITIONED DISH MEDIUM MEDIUM OF RENCA MEDIUM OF MMT 5 DAYS OLD 1   6 ×10⁵   5 × 10⁵   8 × 10⁴ 2 6.8 × 10⁵ 4.2 × 10⁵ 8.5 × 10⁴ 8 DAYS OLD 3 2.8× 10⁶   2 × 10⁶   8 × 10⁴

[0063] When these results are compared to those in example 6, supra, itwill be seen that, while the frozen/thawed RENCA conditioned medium didnot suppress proliferation to the same extent that frozen/thawed MMTconditioned medium did (compare examples 6 and 7), it did, nonetheless,suppress proliferation.

EXAMPLE 11

[0064] The experiments set forth supra showed that frozen conditionedmedium from RENCA- or MMT-containing macrobeads inhibits theproliferation of RENCA cells in vitro. The experiments set forth hereinexamined whether RENCA- or MMT-macrobead conditioned medium, prepared as30 kd or 50 kd concentrates by filtration, would inhibit theproliferation of RENCA cells in vitro. The effects of macrobeadconditioned media were compared to the effects of media conditioned inthe presence of unrestricted RENCA and MMT cells growing in monolayercultures, to determine whether unrestricted tumor cells grown toconfluence also make proliferation regulating material.

[0065] For these experiments, 10 macrobeads, each containing 120,000RENCA or MMT cells (i.e., 1.2×10⁶ cells total) were used to conditionthe medium (complete RPMI) over a period of 5 days. In parallel, 1.2×10⁶RENCA or MMT cells, i.e., the same number of cells, were plated in aculture dish and allowed to proliferate as a monolayer over a period of4 days in complete RPMI medium. Medium was then changed, and this mediumwas collected twenty-four hours later. The reason for the differentlength of time of exposure of the beads and unrestricted cells was thedifference in cell numbers in the monolayers vs. the beads (3- to 5-foldmore cells in the monolayers) at the end of the 5-day period. In otherwords, unrestricted cells grew so much more rapidly than encapsulatedcells, that there were 3-5 times more cells.

[0066] 30 kd and 50 kd filters were used to prepare concentrates of theconditioned media that would, presumably, contain the active material,and would also eliminate toxic metabolic and/or waste materials asconfounding factors in the experiments. These contaminants, which arewell known, are too small to be retained on a 30 kd filter. Filtrateswere also tested, but any interpretation of the results with thismaterial is complicated by the presence of the cellular waste products.A serum-free medium (AIM V) was also used in some experiments to becertain that any effects of serum per se were controlled.

[0067] Essentially, conditioned medium was collected, either three tofive days after the macrobeads had been added to it, or twenty-fourhours after new medium had been added to the unrestricted cells. Themedium was then placed in a test tube filter with an appropriate filter(either a 30 kd or 50 kd filter), and centrifuged for 90 minutes.Material which remained on the filter is referred to as the“concentrate,” while that which spins through the filter and collects atthe bottom of the tube is the filtrate.

[0068] The results, summarized in the Table 6 which follow, show thatwhen the conditioned medium resulting from the restricted RENCA cells inthe macrobeads was used, this inhibited RENCA cell proliferation byabout 52% in two separate experiments. The 50 kd concentrate inhibitedproliferation by about 99%, in both cases, while the 30 kd concentrateinhibited proliferation by about 97%. TABLE 6 Inhibition of RENCA CellGrowth in RENCA Macrobead Conditioned Medium and ReconstitutedConcentrates Unconditioned RENCA Macrobead 30K Concentrate of 50KConcentrate of Plate RPMI Medium Conditioned Medium this Medium thisMedium Number # of Cells # of Cells Inhibition # of Cells Inhibition #of Cells Inhibition 1  1.6 × 10⁶ 7.8 × 10⁵ 51.3% 4.2 × 10⁴ 97% 2.0 × 10⁴99% 2 1.65 × 10⁶ 8.0 × 10⁵ 51.5% 5.0 × 10⁴ 97% 2.0 × 10⁴ 99%

[0069] TABLE 7 Inhibition of RENCA Cell Growth in RENCA Cell CultureConditioned Medium and Reconstituted Concentrates Unconditioned RENCACell Culture 30K Concentrate of 50K Concentrate of Plate MediumConditioned Medium this Medium this Medium Number # of Cells # of CellsInhibition # of Cells Inhibition # of Cells Inhibition 1 1.6 × 10⁶ 1.3 ×10⁶ 18.8% 1.1 × 10⁶ 31.3% 9.0 × 10⁵ 43.8% 2 1.6 × 10⁶ 1.2 × 10⁶ 25.0%1.0 × 10⁶ 37.5% 9.5 × 10⁵ 40.6%

[0070] TABLE 8 Inhibition of RENCA Cell Growth in RENCA MacrobeadConditioned Medium and Concentrate (AIM V Medium) AIM V CONDITIONED 30K50K PLATE CONTROL MEDIUM CONCENTRATE CONCENTRATE NUMBER MEDIUM # cells %inhibition # cells % inhibition # cells % inhibition 1 1.3 × 10⁶ 6.0 ×10⁵ 54% ˜5.0 × 10⁴ 96% ˜4.0 × 10⁴ 97% 2 1.3 × 10⁶ 5.5 × 10⁵ 58% ˜5.0 ×10⁴ 96% ˜4.0 × 10⁴ 97%

[0071] An important point of the experiment is that MMT cells and RENCAcells, when entrapped and restricted in the macrobeads both suppressRENCA cell proliferation, indicating that the proliferation-restrictiveeffect is not specific to tumor type. These experiments confirm those ofExample 8 in which MMT-containing macrobeads suppressed theproliferation of RENCA cells in vivo. In addition, they extend thefindings to indicate that the material released from the macrobeads intothe medium contains molecules that are at least 30 kd in molecularweight which are responsible, in part, for the proliferation-restrictiveeffect. Finally, these experiments show that the macrobead-restrictedRENCA and MMT cells produce far more of the proliferation-suppressingmaterial than the same cells grown to confluency in monolayer cultures.

EXAMPLE 12

[0072] The experiments set forth above show that both MMT- andRENCA-macrobead conditioned media contain material released from theproliferation-restricted cells in the macrobead that can inhibit theproliferation of RENCA cells in vivo and in vitro. Importantly, theexperiments show that the proliferation-inhibitory effect is notspecific to tumor type. The experiments set forth herein examine whetherthe effect is also independent of the species in which the tumororiginally arose. Here, the tumor cell proliferation-inhibitory effectsof a human breast cancer-derived cell line on RENCA cells (usingmacrobeads and macrobead-conditioned media) and also MMT cells (usingmacrobead-conditioned media only) in vitro were examined.

[0073] The methodologies for these in vitro studies were similar tothose described in the examples above. 100,000 MCF-7 cells, (humanbreast cancer cells) were encapsulated in macrobeads, and the resultingMCF-7 macrobeads were incubated with RENCA cells (10,000 per well) for 5days to evaluate the proliferation-inhibitory effects of the macrobeads.In addition, MCF-7 macrobead-conditioned medium was prepared over a5-day incubation period and tested on both RENCA and MMT cells. Cellproliferation was measured over a 5-day period.

[0074] The results are set forth below: TABLE 9 RESULTS OF MCF-7MACROBEADS ON RENCA TARGET CELLS CONTROL Well # (Empty Macrobeads) MCF-7MACROBEADS 1 8.4 × 10⁵ 4.4 × 10⁵ 2 8.0 × 10⁵ 4.4 × 10⁵ 3 7.4 × 10⁵ 3.8 ×10⁵

[0075] TABLE 10 RESULTS OF MCF-7 CONDITIONED MEDIUM ON RENCA TARGETCELLS RPMI Control RPMI Conditioned Plate Medium Medium MCF-7 1 9.0 ×10⁵ 5.0 × 10⁵ 2 8.8 × 10⁵ 4.8 × 10⁵

[0076] TABLE 11 RESULTS OF MCF-7 CONDITIONED MEDIUM ON MMT TARGET CELLSRPMI Control RPMI Conditioned Plate Medium Medium: MCF-7 1 5.0 × 10⁵ 1.5× 10⁵ 2 6.0 × 10⁵ 1.8 × 10⁵

[0077] The results show that MCF-7, a human breast adenocarcinoma cellline, when proliferation-restricted in macrobeads, produces a materialthat inhibits the proliferation of mouse renal adenocarcinoma cells andmouse breast cancer tumor cells to a significant degree (30-70%) asdemonstrated by both the macrobeads themselves and conditioned mediaderived therefrom. This indicates that the proliferation-inhibitoryeffect of growth-restricted cancer cells is independent of both tumortype and species of tumor origin, i.e., mouse and human.

EXAMPLE 13

[0078] The experiments set forth above demonstrate that a human-derivedbreast adenocarcinoma cell line (MCF-7), when growth-restricted inmacrobeads, produces proliferation inhibition of mouse renal and mousebreast adenocarcinoma cells in vitro. The experiments set forth hereinexamine whether a parallel effect of MCF-7-containing macrobeads onRENCA cell tumor growth in vivo exists.

[0079] Eighteen Balb/c mice were injected with 20,000 RENCA cellsintraperitoneally. After three days the mice were divided intotwogroups. Group 1 had six mice and Group 2 had the remaining twelvemice. Group 1 mice, the controls, were transplanted with three emptymacrobeads each. Group 2 received three MCF-7-containing macrobeads(100,000 cells per bead). After twenty-five days, 2 mice from Group 1and three mice from Group 2 were sacrificed. The same number weresacrificed on day twenty-six and the remaining mice were sacrificed onday twenty-seven.

[0080] On necroscopy, the peritoneal cavities of the control mice wereobserved to be completely packed with tumor, and the normal organs weredifficult to identify. We classified this as ++++(100%) tumor intensity.In the treated mice, tumor intensity was rated at +(10-20%).

[0081] These results show that macrobeads containing human breastadenocarcinoma cells are capable of inhibiting renal cell adenocarcinomatumor growth in mice, confirming again that the cancer-cellproliferation/tumor growth-inhibitory effect is neither type-specificnor species-specific.

EXAMPLE 14

[0082] The experiments set forth above demonstrate that the cellproliferation/tumor growth inhibitory effect of macrobeadgrowth-restricted tumors is neither tumor-type nor species specific. Theexperiments set forth herein examine whether (macrobead)proliferation-restricted mouse breast adenocarcinoma cells can inhibitthe growth of both spontaneous mammary tumors and tumors resulting fromthe injection of MMT cells.

[0083] C3H mice have a very high incidence of the development of mammarytumors over their life span. Seven mice at risk for the development ofsuch tumors showed tumors at sixteen months of age. At this time, fiveof the seven mice were implanted with four MMT macrobeads containing100,000 cells each. The remaining two control mice received four emptymacrobeads each. The two control mice developed large tumors and diedwithin three months after the bead implants. The treated mice weresacrificed eleven months after the MMT macrobead implants. The retrievedmacrobeads, organs and tumors were examined grossly and histologically.Hemotoxylin & Eosin staining of the MMT macrobeads showed viable cells.The pre-existing tumors had not increased in size, and there was noevidence of any new tumor development.

[0084] Experiments in which MMT tumor cells were injected subcutaneouslyin the thoracic region were also performed. Fourteen C3H mice weredivided into two groups. The five control group mice were implanted withthree empty macrobeads each. The nine treated mice received threeMMT-containing macrobeads (240,000 cells each). Three weeks afterimplantation all fourteen mice were injected subcutaneously in themammary area with 20,000 MMT cells each.

[0085] Within twenty-five to thirty days, the five control group micebecame ill with evident tumor formation, and all were dead bythirty-five days post-injection. The nine treated mice, observed weekly,continued without any evidence of tumor formation or ill health duringthis period. Ten to twelve months after tumor injection, four of thenine treated mice developed lumps and lost their fur in patches. Theremaining five mice were implanted again with three MMT macrobeadsthirteen months after the initial tumor injection. One mouse died threedays after this surgery, but on necropsy was completely free of tumor.The four surviving mice were sacrificed eight months after the secondmacrobead implant. Necropsy showed minimal or no tumor proliferation.

[0086] An additional observation from these experiments was that thebeads retrieved from the first implantation contained viable tumor cellsbased both on histology and their ability to resume aggressive tumorgrowth patterns in tissue culture after removal from the bead.

[0087] The results of these experiments show that the cellproliferation/tumor growth-inhibiting effects of macrobead-restrictedcancer cells, in this case mouse mammary adenocarcinoma cells, caninfluence the development and growth of both spontaneously arisingtumors and experimentally induced tumors arising from the injection oftumor cells into the mammary area.

EXAMPLE 15

[0088] The experiments set forth above demonstrate a tumor cellproliferation/tumor growth-inhibitory effect of macrobeadproliferation-restricted cancer cells that is characterized by itseffectiveness across tumor types and across species, as well as in bothspontaneous and artificially-induced tumors. The experiments describedherein extend these findings to examine the effects ofmacrobead-entrapped, proliferation-restricted human prostateadenocarcinoma-derived cells (ARCap10), mouse (Balb/c) renaladenocarcinoma cells (RENCA cells), and mouse (C3H) mammaryadenocarcinoma cells (MMT) on the proliferation of ARCaP10 tumor cellsand ARCaP10 tumor growth in nude (Nu/Nu) mice.

[0089] In the first series of experiments, fifteen Nu/Nu mice wereinjected with 2.5×10⁶ ARCaP10 cells subcutaneously in the flank. On thetwentieth day after injection, at which time the average maximal tumordiameter was 0.5 cm, the mice were divided into two groups. Nine wereimplanted with four ARCaP10 macrobeads (1.0×10⁵ cells per macrobead)each, and six control mice received four empty macrobeads each.

[0090] Ten weeks after implantation, five of the control mice had verylarge vascularized tumors (average 2.5 cm in diameter) and one mouseshowed a slightly smaller tumor (less than 0.5 cm). In the treatedgroup, five mice showed complete regression of the initial tumors, andall remained tumor free until sacrifice at eight months. Two mice showedno tumor growth, i.e., their tumors had the same maximal diameter asthey had had at the time of implantation of the macrobeads, and two miceshowed tumors that had enlarged since implantation of the macrobeads.

[0091] The results (tumor volume and size (l×w×h)) of an experiment inwhich RENCA-containing macrobeads (1.2×10⁵) were implanted eighteen daysafter subcutaneous flank injection of 3.0×10⁶ ARCaP10 tumor cells peranimal in 4 Nu/Nu mice are set forth below: TABLE 12 SIZE OF TUMORSOBSERVED IN TREATED MICE (in mm) 10 Days 14 Days Treated 3 Days BeforeDay of 3 Days After 6 Days After After After Mouse Transplant TransplantTransplant Transplant Transplant Transplant Number (Mar. 3, 1998) (Mar.6, 1998) (Mar. 9, 1998) (Mar. 12, 1998) (Mar. 16, 1998) (Mar. 20, 1998)1 3.5 × 3 × flat 6.2 × 5.4 × flat 4 × 4 × flat disappearing 0 0 2   3 ×3 × 1.5 5.1 × 2.2 × 2 4 × 2 × 0.5 3 × 3 × 0.4 2 × 2 × 0.3 2 × 2 × 0.3 3  3 × 2.5 × 1 3.1 × 3.3 × 1 3 × 2 × 0.5 3 × 2 × 0.2 3 × 2 × 0.2 3 × 2 ×0.2 4 2.5 × 2.5 × flat 3.2 × 3.4 × 0.5 speck under skin 0 0 0

[0092] TABLE 13 VOLUME OF TUMORS OBSERVED IN TREATED MICE 3 Days BeforeDay of 3 Days After 6 Days After 10 Days After 14 Days After TreatedMouse Transplant Transplant Transplant Transplant Transplant TransplantNumber (Mar. 3, 1998) (Mar. 6, 1998) (Mar. 9, 1998) (Mar. 12, 1998)(Mar. 16, 1998) (Mar. 20, 1998) 1 2.76 8.81 1.68 0 0 0 2 7.10 11.81 2.101.89 0.63 0.63 3 3.95 5.38 1.58 0.63 0.63 0.63 4 1.64 2.86 0 0 0 0

[0093] In another experiment 10 Nu/Nu mice were injected with2.5×10⁶APCaP10 cells, with six of the mice showing tumor developmentsixty-four days after injection. Three of these mice were given four MMTmacrobeads (2.4×10⁵ cells each) and three received empty macrobeads. Theresults are set forth below: TABLE 14 SIZE OF TUMORS OBSERVED IN TREATEDMICE (in mm) 5 Days Before Day of 18 Days After 22 Days After 27 DaysAfter 30 Days After Treated Mouse Transplant Transplant TransplantTransplant Transplant Transplant Number (Feb. 5, 1998) (Feb. 10, 1998)(Feb. 28, 1998) (Mar. 4, 1998) (Mar. 9, 1998) (Mar. 12, 1998) 1 2 × 2 ×1 3 × 3 × 1.5 1 × 1 × 0.5 0 0 0 2 3 × 2 × 1 3 × 2.5 × 1 2 × 2 × flat <1mm <0.8 mm <0.8 mm 3 4 × 4 × 1.5 6 × 6 × 1.5 6 × 2 × flat 4 × 1 × flat 3× 1 × flat 3 × 1 × flat

[0094] TABLE 15 SIZE OF TUMORS OBSERVED IN CONTROL MICE (in mm) Control5 Days Before 18 Days After 22 Days After 27 Days After 30 Days AfterMouse Transplant Day of Transplant Transplant Transplant TransplantTransplant Number (Feb. 5, 1998) (Feb. 10, 1998) (Feb. 28, 1998) (Mar.4, 1998) (Mar. 9, 1998) (Mar. 12, 1998) 1 4 × 4 × 1.5 5 × 5 × 2 6.5 × 6× 3 6.5 × 6 × 3 6.5 × 6 × 3   7 × 7 × 3 2 3 × 2 × 1 4 × 6 × 3 4.5 × 7 ×3   5 × 8 × 3  11 × 12 × 5 13.3 × 13.3 × 6.5 2^(nd) tumor: 6 × 6 × 1 3 5× 4 × 1 5 × 4 × 2   5 × 4.6 × 2.5   5 × 5 × 2.5   6 × 6 × 2.5   7 × 7 ×2.5 (multilobe) 2^(nd) tumor: 2^(nd) tumor:   2 × 2 × 1   3 × 3 × 0.5

[0095] The results of these experiments further confirm thecross-species, cross-tumor nature of the tumor growth-inhibiting effectof proliferation restriction on tumors of various types. In addition,these experiments demonstrate the ability of theproliferation-restricted cancer cells not only to suppress tumor growthand to prevent tumor formation, but also to cause actual regression ofin vivo tumors.

EXAMPLE 16

[0096] The experiments set forth above showed thatproliferation-restricted cancer cells from several types of tumors andspecies can inhibit the proliferation of the same and different cancercell types in vitro and prevent the formation of both spontaneous andinduced tumors, prevent the growth of tumors, and cause tumors toregress in vivo in an effect that is independent of species and cancertype. The experiment set forth herein describes the extension of thefindings to another species (rabbit) and a rabbit tumor known to havebeen induced virally (VX2).

[0097] In this experiment, a New Zealand White Rabbit (2.5 lbs.) wasinjected intramuscularly in one thigh (two sites) with 0.5 ml of a VX2tumor slurry (characterized as being able to pass through a #26 gaugeneedle) at each site. At 3.5 weeks, a 5 cm×2.5 cm (l×w) tumor hadappeared on the dorsal thigh and two 3 cm-diameter tumors were presenton the ventral thigh. At this point, 211 macrobeads (108 RENCA cellbeads, 63 MMT cell beads, and 40 MCF-7 human breast cancercell-containing beads) were implanted intraperitoneally. Within twodays, the tumor on the dorsal thigh had shrunk by approximately 50%;however, the two ventral tumors did not change. The animal wassacrificed ten days after macrobead implantation. On necropsy, there wasa clear difference between the dorsal and ventral tumors in that theformer was much smaller than it had been at the time of macrobeadimplantation, whereas the two ventral tumors were both hemorrhagic andnecrotic.

[0098] This experiment extends the findings of the effectiveness ofproliferation restriction of various types of cancer cells in relationto the prevention, arrest, and even regression of tumor growth toanother species, the rabbit, adds a tumor of known viral origin to thelist of cancer types, and further supports the cross-tumor andcross-species nature of the growth inhibiting effect, since acombination of mouse renal, mouse breast and human breast cancercell-containing macrobeads were used. In addition, the experiment adds alarger animal model to the in vivo testing of the effectiveness ofproliferation-restriction of cancer cells for the treatment of cancer.

EXAMPLE 17

[0099] The experiments set forth above show thatproliferation-restriction of various types of tumor cells results intheir ability to inhibit the growth of cells of the same or differenttype in vitro and to prevent the formation of, suppress the growth of,or cause regression of various types of tumors in vivo and that theeffects seen are independent of tumor type and species. The experimentsset forth herein evaluated the long-term viability of theproliferation-restricted RENCA cancer cells in agarose-agarosemacrobeads maintained in culture over periods of 1 month, 6 months, 2years, and 3 years using histological, culture, and in vivo techniques.MMT-containing macrobeads were maintained in culture for up to sixmonths. In addition, RENCA- and MMT-containing macrobeads retrieved fromBalb/c and C3H mice respectively after periods of 2 to 8 months afterimplantation were examined for viable tumor cells by both histologicaland culture techniques.

[0100] For these experiments the agarose-agarose macrobeads wereprepared with either 1.2×10⁵ RENCA cells or 2.4×10⁵ MMT cells. They wereexamined histologically (hermatoxylin & eosin staining) and by culturetechniques for cell viability and tumor characteristics at the intervalsdescribed supra. For the RENCA macrobeads, cell numbers increasedapproximately 3- to 5-fold over the first month with a subsequentadditional doubling in six months. After one year, there was a continuedincrease in cellular mass, but the rate of cell proliferation haddecreased. After two years, amorphous material had begun to appear inthe center of the bead, and the cell mass/numbers did not appear to beincreasing, although mitotic figures are still evident. After threeyears, there appeared to be somewhat more amorphous material in thecenter of the bead, but the cell mass/number was stable. MMT macrobeadshave been followed for only six months, but the early pattern of cellproliferation and bead appearance is similar to that of RENCA.

[0101] For evaluation of the viability and biological behavior of theRENCA and MMT cells at the intervals described above, ten beads werecrushed and plated in two or more 25 cm² tissue culture flasks incomplete RPMI medium. The flasks were then observed for cell growth. Atone and six month intervals, the number of viable cells retrievable fromthe beads increases. At one year, the number of RENCA cells growing fromthe crushed bead appears to be similar to that at six months. At two andthree years, the proportion of viable cells appears to be somewhat less,dropping to approximately 20% of the maximum number they reached in thebead (i.e., in their restricted state) after three years in culture.

[0102] For the evaluation of the retrieved RENCA and MMT macrobeadsafter in vivo implantation (periods of 1-4 years for RENCA macrobeadsand up to 8 months for MMT macrobeads), histological techniques havebeen utilized to date. The patterns of cell proliferation and mass arevery similar to those of the beads maintained in culture for thecorresponding periods of time, i.e., the cells increase in number atleast up to 4 months for RENCA and 8 months for MMT.

[0103] For the other cancer cell lines-with which we have been working,such as MCF-7 and ARCaP10, the viability patterns in macrobeads aresimilar to those observed for RENCA and MMT.

[0104] These experiments show that cancer cells can be maintained invitro for periods of up to 3 years and in vivo for periods of at least 8months in a proliferation-restricting environment and that they maintaintheir viability for these periods with clear demonstration of increasingcell numbers up to at least one year. This is important not only for theability to create and store cancer treatment materials, but also for theability of the proliferation-restricted cells to put out tumor growthsuppressing material in warm-blooded animals over the continuous,prolonged periods likely to be necessary for the successful treatment ofexperimental or naturally-occurring cancer.

EXAMPLE 18

[0105] The experiments set forth above show that cancer cells of varioustypes can be maintained under proliferation-restricted conditions forlong periods of time (up to 3 years) with retention of their ability toproliferate, form tumors, and release cell-proliferation-inhibiting andtumor-growth preventing, suppressing, and even regressive materials. Theexperiments set forth herein evaluate the possible toxicity of long-term(one-year) implants of cancer cell-containing, agarose-agarosemacrobeads in Balb/c mice.

[0106] Seven Balb/c mice were implanted with 3 RENCA macrobeads each(1.2×10⁵ cells per bead). Immediately after surgery the mice appearedill (spiky fur and lethargy) for a few days, but became healthy againafter this. All mice survived in apparent good health for a period of atleast one year, with one mouse dying of old age and another of unrelatedcauses. All mice were sacrificed. On necropsy, no abnormalities, such asfibrosis, peritonitis, or tumor growth were observed. All organsobserved appeared normal, although some adherence of the beads to theserosal surfaces of the intestines were observed, especially where therewere intestinal loops. No interference with the normal function orstructure of the intestines has been observed.

[0107] These results show that cancer cell-containing agarose-agarosemacrobeads are well tolerated in experimental animals over a one-yearperiod. These findings show that the proliferation-restrictingcancer-cell beads can be utilized in vivo for the prevention,suppression and regression of the growth of in vivo tumors of varioustypes.

[0108] The foregoing examples describe the invention, which includes,inter alia, compositions of matter which can be used to produce materialwhich suppresses proliferation of cancer. These compositions comprisecancer cells entrapped in a selectively-permeable material to form astructure which restricts the proliferation of the entrapped cells. As aresult of their being restricted, the cells produce unexpectedly highamounts of material which suppresses proliferation of cancer cells. Therestricted cells produce more of the material than comparable,non-restricted cancer cells.

[0109] The matter used to make the structures of the invention includeany biocompatible matter which restricts the growth of cancer cells,thereby inducing them to produce greater amounts of cancer cellproliferation/tumor growth-suppressing material. The structure has asuitable pore size such that the above material can diffuse to theexternal environment, and prevent products or cells from the immunesystem of the host from-entering the structure and causing the rejectionof or otherwise impair their ability to survive and continue to producethe desired material. The matter used to form the structure will also becapable of maintaining viable (proliferation-restricted, but surviving)cells both in vitro and in vivo, preferably for periods of up to severalyears by providing for the entrance of proper nutrients, the eliminationof cellular waste products, and a compatible physico-chemicalintra-structural environment. The matter used to prepare the structureis preferably well tolerated when implanted in vivo, most preferably forthe entire duration of implantation in the host.

[0110] A non-limiting list of materials and combinations of materialsthat might be utilized includes alginate-poly-(L-lysine);alginate-poly-(L-lysine)-alginate;alginate-poly-(L-lysine)-polyethyleneimine; chitosan-alginate;polyhydroxylethyl-methacrylate-methyl methacrylate;carbonylmethylcellulose; K-carrageenan; chitosan;agarose-polyethersulphone-hexadi-methirine-bromide (Polybrene);ethyl-cellulose; silica gels; and combinations thereof.

[0111] The structures which comprise the compositions of matter may takemany shapes, such as a bead, a sphere, a cylinder, a capsule, a sheet orany other shape which is suitable for implantation in a subject, and/orculture in an in vitro milieu. The size of the structure can vary,depending upon its eventual use, as will be clear to the skilledartisan.

[0112] The structures of the invention are selectively permeable, suchthat nutrients may enter the structure, and so that theproliferation-inhibiting material as well as cellular waste may leavethe structure. For in vivo use, it is preferred that the structuresprevent the entry of products or cells of the immune system of a hostwhich would cause the rejection of the cancer cells, or otherwise impairtheir ability of the cancer cells producing theproliferation-suppressive material.

[0113] Another aspect of the invention includes compositions which areuseful in suppressing cancer cell proliferation. These compositions areprepared by culturing restricted cells as described supra in anappropriate culture medium, followed by recovery of the resultantconditioned medium. Concentrates can then be formed from the conditionedmedium, e.g., by separating fractions having molecular weight of greaterthan 30 kd or greater than 50 kd, which have high anti-proliferativeeffect on cancer cells.

[0114] As the examples show, the invention is not limited to anyparticular type of cancer; any neoplastic cell may be used in accordancewith the invention. Exemplary types of cancer cells which can be usedare renal cancer cells, mammary cancer cells, prostate cancer cells,choriocarcinoma cells and so forth. The cancer cells may be ofepithelial, mesothelial, endothelial or germ cell origin, and includecancer cells that generally do not form solid tumors such as leukemiacells.

[0115] As will be clear from this disclosure, a further aspect of theinvention is therapeutic methods for treating individuals suffering fromcancer. When used in a therapeutic context, as will be elaborated uponinfra, the type of cancer cell restricted in the structure need not bethe same type of cancer from which the subject is suffering, although itcan be. One such method involves inserting at least one of thestructures of the invention into the subject, in an amount sufficient tocause suppression of cancer-cell proliferation in the subject.Preferably, the subject is a human being, although it is applicable toother animals, such as domestic animals, farm animals, or any type ofanimal which suffers from cancer.

[0116] The composition of the present invention can be used as primarytherapy in the treatment of cancer, and as an adjunct treatment incombination with other cancer therapies. For example, patients may betreated with compositions and methods described herein, in conjunctionwith radiation therapy, chemotherapy, treatment with other biologicallyactive materials such as cytokines, anti-sense molecules, steroidhormones, gene therapy, and the like. Additionally, the compositions andmethods of the invention can be used in conjunction with surgicalprocedures to treat cancer, e.g., by implanting the macrobeads afterresection of a tumor to prevent regrowth and metastases. Cancers whichpresent in an inoperable state may be rendered operable by treatmentwith the anti-proliferative compositions of the invention.

[0117] The compositions of the invention can also be usedprophylactically in individuals at risk for developing cancer, e.g.,presence of individual risk factors, family history of cancer generally,family history of cancer of a specific type (e.g. breast cancer), andexposure to occupational or other carcinogens or cancer promotingagents. For prophylaxis against cancer, a prophylactically effectiveamount of the structures of the invention are administered to theindividual upon identification of one or more risk factors.

[0118] As indicated by the examples, supra, the antiproliferative effectis not limited by the type of cancer cell used, nor by the species fromwhich the cancer cell originated. Hence, one can administer structureswhich contain cancer cells of a first type to a subject with a second,different type of cancer. Further, cancer cells of a species differentfrom the species being treated can be used in the administeredstructures. For example, mouse cancer cells may be restricted in thestructures of the invention, and then be administered to a human. Ofcourse, the structures may contain cancer cells from the same species asis being treated. Still further, the cancer cells may be taken from theindividual to be treated, entrapped and restricted, and thenadministered to the same individual.

[0119] Yet another aspect of the invention is the use of concentrates,as described herein, as a therapeutic agent. These concentrates may beprepared as described herein, and then be administered to a subject withcancer. All of the embodiments described supra may be used in preparingthe concentrates. For example, following in vitro culture of structurescontaining mouse cancer cells, concentrates can be prepared and thenadministered to humans. Similarly, the structures can contain humancells, and even cells from the same individual. Also, as discussedsupra, the type of cancer cell used to prepare the concentrate may be,but need not be, the same type of cancer as the subject suffers from.Hence, murine mammary cancer cells may be used, e.g., to prepare aconcentrate to be used to treat a human with melanoma, or an individualwith prostate cancer may have some of his prostate cancer cells removed,entrapped in a structure of the invention, cultured in an appropriatemedium, and then have resulting conditioned medium filtered to produce aconcentrate. It should be borne in mind that the conditioned mediaresulting from in vitro cultures of the structures of the invention isalso a part of the invention.

[0120] Processes for making the structures of the invention, as well asthe concentrates of the invention, are also a part of the invention. Inthe case of the concentrates, one simply cultures the structures of theinvention for a time sufficient to produce a sufficient amount ofantiproliferative material and then separates the desired portions fromthe resultant conditioned medium, e.g., by filtration with a filterhaving an appropriate cut off point, such as 30 kilodaltons or 50kilodaltons.

[0121] Other facets of the invention will be clear to the skilledartisan, and need not be set out here.

[0122] The terms and expression which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

We claim:
 1. Composition useful in suppressing proliferation of targetcancer cells, produced by entrapping a sample of cancer cells in abiocompatible, proliferation-restrictive, selectively-permeablestructure, culturing said structure in culture medium for a timesufficient to restrict the proliferation of said entrapped cancer cells,wherein said entrapped cancer cells produce cancer-cell proliferationsuppressing material which suppresses proliferation of the target cancercells and recovering the medium.
 2. The composition of claim 1, whereinsaid filter separates material having a molecular weight of at leastabout 30 kd from material having a molecular weight less than about 30kd, wherein said composition comprises material having a molecularweight of at least about 30 kd.
 3. The composition of claim 1, whereinsaid entrapped cancer cells are of epithelial origin.
 4. The compositionof claim 1, wherein said entrapped cancer cells are breast cancer cells,renal cancer cells, prostate cancer cells or choriocarcinoma cells. 5.The composition of claim 1, wherein said entrapped cancer cells arehuman cancer cells.
 6. The composition of claim 1, wherein saidentrapped cancer cells are mouse cancer cells.
 7. The composition ofclaim 1, wherein said structure contains from about 10,000 to about500,000 cancer cells.
 8. The composition of claim 1, wherein saidstructure contains from about 30,000 to about 250,000 cancer cells.
 9. Aprocess for making a composition which has a cancer cellproliferation-inhibiting effect, comprising culturing a biocompatible,proliferation-restrictive, selectively-permeable structure which hascancer cells entrapped therein in a medium for a time sufficient forsaid cancer cells to produce cancer-cell proliferation-suppressingmaterial, and recovering the medium.
 10. The process of claim 9,comprising filtering said medium through a filter which retains materialhaving a molecular weight of at least about 30 kd.
 11. The process ofclaim 9, wherein said medium is serum free.
 12. The process of claim 9,wherein said cancer cells are human cancer cells.
 13. The process ofclaim 9, wherein said cancer cells are mouse cancer cells.
 14. Theprocess of claim 9, wherein said cancer cells are of epithelial origin.15. The process of claim 14, wherein said cancer cells are selected fromthe group consisting of breast cancer cells, renal cancer cells,prostate cancer cells, and choriocarcinoma cells.
 16. The process ofclaim 9, wherein said structure contains from about 10,000 to about500,000 cells.
 17. The process of claim 16, wherein said structurecontains from about 30,000 to about 250,000 cells.
 18. The process ofclaim 9, wherein said structure is a bead.
 19. A method for suppressingthe proliferation of cancer cells in a subject in need thereof,comprising administering to said subject a therapeutically effectiveamount of biocompatible, proliferation-restrictive,selectively-permeable structures which contain restricted cancer cellsfrom a species other than said subject, wherein said restricted cancercells produce a material which suppresses cancer cell proliferation, inan amount sufficient to suppress cancer cell proliferation in saidsubject.
 20. The method of claim 19, wherein said restricted cancercells are a type of cancer different from the type of cancer with whichsaid subject is afflicted.
 21. The method of claim 19, wherein saidrestricted cancer cells are the same type of cancer with which saidsubject is afflicted.
 22. The method of claim 19, wherein said structureis a bead.
 23. The method of claim 19, wherein said structure containsfrom about 10,000 to about 500,000 cells.
 24. The method of claim 23,wherein said structure contains from about 30,000 to about 250,000cells.
 25. The method of claim 19, wherein said restricted cancer cellsare of epithelial origin.
 26. The method of claim 25, wherein saidrestricted cancer cells are selected from the group consisting of breastcancer cells, prostrate cancer cells, renal cancer cells, andchorio-carcinoma cancer cells.
 27. The method of claim 19, wherein saidsubject is a human.
 28. The method of claim 27, wherein said cancercells are mouse cancer cells.
 29. A method for suppressing proliferationof cancer cells in a subject in need thereof, comprising administeringto said subject a therapeutically effective amount of biocompatible,proliferation-restrictive, selectively-permeable structures whichcontain restricted cancer cells from the same species as said subject,wherein said restricted cancer cells produce a material which suppressescancer-cell proliferation, in an amount sufficient to suppresscancer-cell proliferation in said subject.
 30. The method of claim 29,wherein said restricted cancer cells are from a different individualthan said subject.
 31. The method of claim 29, wherein said restrictedcancer cells are taken from the subject to which said structures areadministered.
 32. The method of claim 29, wherein said subject is ahuman.
 33. The method of claim 29, wherein said structure is a bead. 34.The method of claim 29, wherein said restricted cancer cells are ofepithelial origin.
 35. The method of claim 29, wherein said restrictedcancer cells are of a type different from the cancer with which saidsubject is afflicted.
 36. The method of claim 29, wherein saidrestricted cancer cells are of the same type as the cancer with whichsaid subject is afflicted.
 37. The method of claim 34, wherein saidrestricted cancer cells are breast cancer cells, renal cancer cells, orprostate cancer cells.
 38. The method of claim 29, wherein saidstructure contains from about 10,000 to about 500,000 cells.
 39. Themethod of claim 38, wherein said structure contains from about 30,000 toabout 250,000 cells.
 40. A method for suppressing proliferation ofcancer cells in a subject in need thereof, comprising administering anamount of the composition of claim 1 to said subject in an amountsufficient to suppress proliferation of cancer cells in said subject.41. The method of claim 40, wherein said subject is a human.
 42. Themethod of claim 41, wherein said entrapped cancer cells are not humancells.
 43. The method of claim 42, wherein said entrapped cancer cellsare mouse cells.
 44. The method of claim 41, wherein said entrappedcancer cells are human cells.
 45. The method of claim 40, wherein saidrestricted cancer cells are of the same type as the cancer with whichsaid subject is afflicted.
 46. The method of claim 40, wherein saidrestricted cancer cells are cancer cells taken from the subject to whichsaid structure is administered.
 47. The method of claim 40, wherein saidrestricted cancer cells are of epithelial origin.
 48. The method ofclaim 47, wherein said restricted cancer cells are selected from thegroup consisting of renal cancer, choriocarcinoma, breast cancer, andprostate cancer.
 49. The method of claim 40, wherein said structurecontains from about 10,000 to about 500,000 cells.
 50. The method ofclaim 49, wherein said structure contains from about 30,000 to about250,000 cells.
 51. A composition of matter comprising a biocompatible,proliferation-restrictive, selectively-permeable structure, saidstructure restricting cancer cells which produce more of a materialwhich suppresses cancer cell proliferation compared to an equal numberof the same cancer cells when unrestricted.
 52. A process for preparinga biocompatible, proliferation-restrictive, selectively-permeablestructure, comprising the steps of forming a structure by contactingcancer cells with biocompatable, proliferation-restrictive matter toform the structure, and culturing the structures for a sufficient periodof time to restrict said cancer cells such that they produce a materialwhich suppresses cancer cell proliferation compared to an equal numberof unrestricted cancer cells of the same cancer type.
 53. A method ofincreasing the production of material that suppresses cancer cell growthby a cancer cell, comprising restricting cancer cells with astructure-forming material to form a biocompatable,selectively-permeable, proliferation-restrictive structure and culturingthe cancer cells until they are restricted.